The overall objective of this renewal grant application is to gain further understanding into the mechanism of coronavirus RNA synthesis, by using mouse hepatitis virus (MHV) as a model system. We will use cloned defective interfering (DI) RNAs and another DI system in which a subgenomic DI RNA is synthesized from an inserted MHV transcription consensus sequence. We established such a system in which a subgenomic RNA is synthesized from an expressed MHV-RNA fragment in MHV-infected cells. Expressed RNA did not replicate. Expression of a complete copy of (-)-stranded DI RNA initiated DI RNA replication and subgenomic DI RNA transcription. We plan to use these cloned MHV DI systems and RNA expression system to study the detailed mechanisms of coronavirus RNA replication and transcription. I. Mechanism of MHV RNA replication: We identified two internal cis-acting replication signals, which are necessary for MHV DI RNA synthesis. By making use of a unique cold- sensitive phenotype of the internal cis-acting replication signals, we can learn how they affect MHV DI RNA replication by: precisely identifying their biologically active sites, analyzing their structures, and determining the strand-specificity of the RNA synthesis that they govern. We will also study the effects of these regions on aspects of RNA stability, protein-RNA interaction, and RNA-RNA interaction. Using (+)- and (-)-stranded RNA expression systems we will identify sequences necessary for copying (-)- and (+)-stranded RNAs. II. Mechanism of coronavirus transcription We will study transcription regulation of the sequences flanking the transcription consensus sequence. We will analyze how the RNA structure between two proximally inserted intergenic regions inhibits transcription from the more upstream intergenic region. We will identify the transcription step that determines the relative amounts of subgenomic mRNAs. We will study whether de novo (+)-sense genomic DI RNA synthesis is necessary for subgenomic DI RNA synthesis. We will also study the effect of secondary transcription on subgenomic DI RNA accumulation. We will identify the sequences necessary for subgenomic RNA synthesis using the (+)- and (-)-strand RNA expression systems. We will also examine the possibility that subgenomic RNA synthesis involves processing of precursor RNA.